1. Field
The invention is in the general field of medical laboratory equipment and is particularly concerned with cytocentrifuges, i.e. apparatus for centrifugally depositing, on microscope slides, cells from a liquid suspension of same and with methods of using such apparatus.
2. State of the Art
Apparatus of the type concerned have been developed heretofore and are widely used. Perhaps the best known cytocentrifuge is the "Cytospin" produced by or under the auspices of Shandon Southern Products Limited, Rancorn, England, as shown by Gordon U.S. Pat. Nos. 4,391,710 of Jul. 5, 1983 and by Griffin 4,678,579 of Jul. 7, 1987. There, as in other apparatus of the type concerned, a liquid suspension of cells to be deposited on a microscope slide is placed in a sample chamber of a cytocentrifuge device, normally one adapted for installation in a centrifuge that is adapted to receive a plurality of such devices, each equipped with a sample chamber, a filter pad holder, and a slide holder, so multiple microscope slides can be prepared at the same time, such liquid suspension of cells being passed by centrifugal force from sample chamber to slide surface through an opening in a filter pad that is clamped against the receiving surface of the slide for both sealing so far as possible against lateral passage and consequent loss of the cell-carrying liquid with its cells and for absorbing the liquid component of such cell-carrying liquid upon deposit of the cells on the slide.
Alan J. Gordon, inventor in the aforesaid U.S. Pat. No. 4,341,710, improved pre-existing machines for Shandon in various respects, primarily by incorporating slide and filter card clamping means as part of a sample chamber unit that is removable unitarily (with clamping means) from a centrifuge rotor of the cytocentrifuge.
Prewetting of the liquid-absorbent filter pad has been resorted to in some instances prior to centrifugation to minimize attraction for the cell-carrying liquid sample as it passes toward the microscope slide, with consequent diversion into the filter pad with loss of cells before they have a chance to adhere to the slide.
Also, utilizing a sample chamber having depth that normally dams against liquid outflow except under centrifuging action has been tried in the past for both centrifugal chemical analyzers and cytocentrifuges, see particularly the N. G. Anderson article entitled "The Development of Fast Analyzers", Z. Anal. Chem. 257-271 (1972), and Wells U.S. Pat. No. 4,428,323 of Jan. 31, 1984, respectively.
Prior to the development of cytocentrifuges utilizing centrifugal force to propel a sample of a cell-carrying liquid toward and onto a microscope slide, application of cells onto microscope slides was accomplished by sedimentation techniques utilizing gravity to flow samples of a cell-carrying liquid onto the slides, see the article in ActaCytologica 8, 234-241 (1964) by G. Th. A. M. Bots et al.
Present day cytocentrifuges do significantly better than the sedimentation technique. This is accomplished by the use of high centrifugal speeds, wherein sedimentation velocity of cells suspended in the cell-carrying liquid onto the slide is considerably greater than the hydraulic force exerted laterally of the slide face on which the cells are to be deposited.
In practice, present systems employ a trade-off between filter pad thickness governing liquid-holding capacity and a conveniently handled clamping force holding the filter pad against the slide during centrifugation. This places practical constraints on the range of performance characteristics of current systems. For example, if a very slow liquid flow rate into the filter pad is desired, with a convenient clamping spring force, a very thin filter pad must be employed. Under these conditions, the quantity of the sample will be limited to the volume of liquid that such filter pad will hold. If a larger quantity of the sample liquid is involved, a relatively thick filter pad should be employed to receive and retain the greater quantity of liquid component of the liquid sample, but then the flow rate can be too fast to allow good cell recovery.
Loss of cells is also suffered in the transition from the stationary state of the loaded sample chamber to the ultimate steady state speed of operation. Such loss is most severe when the sample liquid comes into contact with the filter pad prior to initiation of centrifugation. Lateral flow into the filter without centrifugation takes place as permitted by the clamping force, filter characteristics, and system geometry. With no centrifugal force to sediment the cells against the slide, the cells tend to flow into the filter with the liquid. If a large fraction of the sample flows into the filter under these conditions, a correspondingly large fraction of the cells can be lost. It has therefore been recognized that some means must be provided to prevent premature contact of the liquid sample with the filter pad. Alan J. Gordon (U.S. Pat. No. 4,391,710) accomplished this for Shandon by providing a tilting chamber. John Wells (U.S. Pat. No. 4,428,323) on the other hand provided a well for the sample liquid as a dam against premature release of such sample, as N. G. Anderson had done for the so-called "Fast Analyzer". The force required, and hence the speed of delivery, is determined by the depth of the well and the slope of the dam.
With the above provisions, transitional cell loss is significantly reduced but it still occurs to an undesirable extent. Even with the system of John Wells, the liquid is forced over the dam upon start up and into the flow passage leading therefrom. Cell-carrying sample liquid rushes into the empty flow passage on start up. Since the cells are uniformly suspended in the sample liquid, the initial contact with the filter pad will cause absorption of cell-containing liquid.
Another source of cell loss is during wet fixation after the cells have been collected on the slide. Normally, the collected cells are sprayed with, or the slide is immersed in, an aqueous alcohol fixative solution. Residual liquid on the slide interacts with the fixative solution and, together with the forces involved in applying the solution, often results in dislodging of otherwise adherent cells from the slide.
Some manufacturers recommend addition of fixative liquid into the sample chamber after addition of the cell-containing liquid sample so that cells will be fixed during centrifuging. However, there is no provision for separating the fixative from the sample, and cells become fixed prior to collection on the slide. This results in cells which do not properly flatten and therefore do not adhere well to the slide, thus producing poor morphology and cell loss. No equipment presently exists which provides for in-situ fixation during centrifuging following cell sedimentation.